Abstract: Aim: To investigate cell proliferation and invasiveness of cervical cancer HeLa229 cell after inhibition of the NF-κB signaling pathway by p65siRNA. Methods: RNA interference was employed for specific inhibition of the expression of p65. The HeLa229 cell was divided into transfected group and untransfected group. Cell viability was detected by MTT after the HeLa229 cells were transfected with or without p65siRNA for 24, 48, 72h. The sensitivity to 5-Fu of the HeLa229 cell, transfected with or without p65siRNA, was evaluated also by MTT. Boyden chamber experiment in vitro was used to detect the invasion ability of HeLa229 cell. Results: p65 siRNA inhibited the cell proliferation as compared with the untransfected cells. Proliferations of both cells transfected with and without p65 siRNA were inhibited in a concentration-dependent manner, while at the same concentration of 5-Fu the viability of transfected HeLa229 cells was significantly suppressed (P <0.05). Compared with the untransfected cells, the number of cells which traversed through Matrigel was decreased obviously. Conclusion: RNAi targeting of p65 has anti-proliferative effects, inhibits the invasiveness and increases the 5-Fu sensitivity of the ESCC cells, suggesting that NF-κB might be a good target for cancer treatment.